Fundamental knowledge of the periodontium is required to develop new approaches to understand and treat this major health problem. This project is directed at understanding the role and mechanism of the Bmp2 gene and Sost gene in periodontium function. Our recent published results indicate that the mouse Bmp2 gene in stem-progenitor cells in the periodontium is required for the development of the periodontium and formation of tooth root. Deletion of the Bmp2 gene in mesenchymal stem cells of bone marrow suggests that Bmp2 is a critical factor that activates these stem cells located on the small capillary vascular bed to differentiate toward mature osteoblasts and osteocytes. We now want to test the idea that the Bmp2 gene in stem cells of the periodontium, similar to mesenchymal cells in bone is critical for their lineage progression to the more complex situation of differentiation to cellular and acellular cementum and the tendon-like periodontal ligaments within the periodontium, using state of the art lineage tracing methods in vivo. In this proposal, the Bmp2 gene will be removed from two major classes of stem cells (aSmooth Muscle actin positive or aSMA+ and Osterix+ cells) of the periodontium and determine the role of the Bmp2 gene in specification and differentiation of the PDL and its role in attachment to the bone and teeth. Recently, the function of the Sost gene has been shown to be a negative regulator of cementum and alveolar-basal bone surrounding the teeth. In a model of periodontal degeneration, the Periostin KO deletion of the Sost gene greatly corrects many of the periodontal defects, especially in the alveolar bone, and increases differentiation of PDL progenitors to new alveolar bone. Treatment of mice with Sclerostin antibody (Scl-Ab), in clinical trials for treating osteoporosis, also greatly corrects these periodontal defects in the Periostin KO. We will now cross the Bmp2 cKO model with Sost KO and treat the Bmp2 cKO model with Scl Ab and determine the rescue of the periodontal defects in the Bmp2 cKO model. Using a periodontium stromal cell model, highly enriched in periodontal stem cells, we will delete the Bmp2 gene and determine the gene expression profiles during differentiation using RNA-seq methods and chromatin maps of key markers of promoters and enhancers, and DNase hypersensitive site maps. We will use RNA-seq methods to determine the kinetics of differentiation from stem cell to periodontal-like and mineralized structures. We will isolate primary periodontal cell, from 2 week animals that have no cellular cementum related but acellular cementoblast precursors, and PDL and osteoblast cells, 2 month animals undergoing active cellular cementogenesis, PDL formation and osteogenesis, and 6 month animals in which cementogenesis is complete. We will determine the expression profiles at these 3 stages and determine a set of genes selective for acellular cementoblast and cellular cementoblasts and cementocytes. By validation in vivo, we will determine a set of genes that is selectively expressed during cementogenesis and not osteogenesis. These experiments have high clinical relevance and fundamental basic periodontal biology.